The overall objective of this research is to continue our work using leucine tracer and isotope dilution methods to characterize the hormonal and substrate factors which regulate the metabolism of the essential amino acid leucine with the ultimate goal of making valid extrapolations from the leucine kinetic data to whole body protein metabolism in normal and diabetic humans. These Isotope dilution methods utilizing a variety of leucine tracers are being widely employed by a number of investigators including ourselves, but clear validation of the assumptions involved in the model (s) used to interpret these data from such studies are lacking. On the basis of results generated from the previous funding period, we have developed the "reciprocal pool model" of leucine metabolism in which we hypothesize that the plasma specific activity (SA) of the transaminated product (the reciprocal pool SA) of the infused leucine tracer (e.g. the plasma (3H) alpha-ketoisocaproate, (3H) KIC, during infusion of (3H) leucine or (14C) leucine during infusion of (14C)KIC) accurately reflects the SA (and metabolism) of intracellular leucine. The major focus of this competitive renewal is to attempt to validate further (and identify the limitations of) this model by exploring the following specific aims: 1) to exclude, in humans, significant isotope effects among 3H, 2H, 14C and 13C tracers of leucine and KIC and between H13CO3 and H14CO3; 2) to characterize in vivo the relationship between the plasma reciprocal pool and a) the intracellular free leucine SA, and B) the SA of the intracellular pool(s) from which protein synthesis and KIC oxidation occur: 3) to determine whether (or not) the reciprocal pool model can be validly utilized under nonsteadystate conditions; 4) to determine whether isotope recycling is the result of incorporation and release of tracer from protein and/or the sequestration of tracer in an as yet unidentified pool(s); 5) to determine if protein synthesis is substrate limited under hyperinsulinemic conditions in humans; and 6) to explore the hepatic metabolism of portally infused tracer in an attempt to develop valid models for the study of enterally absorbed amino acids. Completion of these studies will permit a greater understanding of the relationship between leucine metabolism as studied by isotope dilution methods and whole body protein metabolism.